Indicators are materials that produce a detectable signal (also denoted “dS”) in response to an external stimulus (also denoted “eS”). Such stimuli typically include temperature, light (photo-labile or photochromic indicators), electric field (electrochromic indicators), pressure (piezoelectric indicators), ion concentration (e.g. pH indicators) and biochemical reactivity (e.g. enzyme indicators).
The mechanism of translation of stimulus into detectable signal is illustrated in Scheme I and typically involves the chemical removal or modification of a labile group (also denoted “LG”) of the indicator in a process mediated by the experience of said stimulus.

Removal or modification of the labile group most often yields an activated signalogen (also denoted “aS”) which typically undergoes further transformation which often involves interaction with auxiliary reagents (also denoted “aR”) to yield a signalophore (also denoted “SP”), the formation of which usually coincides with the occurrence of said detectable signal. Therefore, an indicator represents an activated signalogen that is masked, i.e. inactivated by a labile group. An indicator system (also denoted “IS”) comprises the elements of activated signalogen, labile group, auxiliary reagent (optional), detectable signal and suitable means for interrogation of detectable signal. The appearance of a detectable signal is a consequence of experiencing an external stimulus and hence allows ready detection and/or quantification of the external stimulus by interrogation with suitable instrumentation or the human eye.
Examples of detectable signal include change in optical density, absorption or emission (e.g. chromogenic, fluorogenic and luminogenic indicators) or change in electric current or potential (electrogenic indicators).
A detectable signal may be transient or persistent in nature: For example a fluorogenic enzyme indicator may release a fluorescent entity upon experience of certain enzymatic activity. In this case the detectable signal is persistent in nature. In contrast a bioluminogenic indicator may emit light in response to the presence of ATP. The emission of light representing the detectable signal is transient in nature.
A detectable signal may be associated with transient or persistent external stimuli: For example, pH indicators continuously change color in response to changing proton concentrations, which represents a persistent external stimulus. In this case the corresponding indicators undergo reversible change and are termed reversible indicator systems. In contrast a photo-labile indicator may undergo lasting color change in response to a short laser pulse. In this case the corresponding indicators undergo irreversible change upon a single experience of external stimulus and are termed irreversible indicator systems.
There exist a large number of important technical applications including biology, diagnostics, chemistry and optical data storage that relate to irreversible indicators.
Indicators used in biology often respond to enzymatic activity. Enzyme indicators are used in many formats of assay. Such assays may be performed in solution phase which requires a soluble indicator system.
Other assays require the physical location of enzymatic activity. Here, a suitable indicator will stain the specific site associated with enzyme activity. For this purpose the indicator must form an insoluble precipitate in response to localized enzymatic stimuli. These types of indicators are termed insoluble or precipitating indicator systems. Precipitating indicator systems by nature are irreversible.
Detection of enzyme activity is important in diagnostic and testing applications as well as in biochemistry, molecular biology and histology research. In diagnostics, enzyme activity relates to the presence of microbial pathogens, as well as to metabolic ill functions or to genetic disorders.
Immunological methods are based on the interaction of antibodies with antigens. In order to detect such interaction, antibodies must be carrying a label. Enzymes are commonly used to label secondary antibodies. Hence, the detection of enzyme activity is fundamental to immunological assays. Immunological assays are widely used in clinical diagnostics, food and environmental testing, as well as in biochemical protocols such as Western blotting.
In molecular biology the detection of enzyme activity is needed in reporter gene protocols. Genetic expression of a reporter gene gives rise to enzyme activity that can be detected. This way, the researcher obtains information on genetic transformations.
1H-Indox-3-yl Indicator Systems
1H-Indox-3-yl (also denoted “Indox”) indicators are well known chromogenic indicators widely used to visualize enzymatic activity in microbiology, immunology, biochemistry and genetics.
Indox indicator systems are derived from the 1H-indol-3-ol (3-hydroxyindol, respectively its tautomeric form indolin-3-one or 3-oxoindoline, denoted Indoxol) structure where the hydrogen atom of the 3-hydroxyl group is replaced by a labile group. Loss of the labile group yields Indoxol as an activated signalogen which interacts with atmospheric oxygen (a common auxiliary reagent) in a complex radical chain reaction to yield colloidal indigo stains as signalophor. A dramatic change in optical transmission of a sample associated with indigo dye formation represents a detectable signal of the indicator system.
Despite their widespread use and commercial significance, applications of Indox indicator systems suffer from some major limitations:
For instance, Indox indicator systems depend on molecular oxygen or other oxidizing auxiliary reagents to develop desired indigo signalophores. Due to said requirement indoxyl substrates are of limited or no use under anaerobic conditions. Considering the portion of enzyme assays that are performed in the absence of oxygen, this limitation is significant.
Therefore, indicators with properties similar to indoxyl indicators without the undesirable dependence on molecular oxygen or other oxidizers would constitute a major improvement over the state of the art.
Most popular Indox indicators yield stains ranging from violet to green because red indigos are less effective for the purpose of staining.
However, it would be desirable to have available a choice of colors when using chromogenic or fluorogenic indicator systems. This is of particular importance in dual or multiple enzyme assays requiring parallel detection or in applications that require optical contrast against off white background.
Therefore, novel chromogenic enzyme indicators that expand the current color selection into the range of yellow to red would represent a further valuable addition to the art.
Moreover, common enzyme indicators are either chromogenic or fluorogenic in nature: For example, common commercial enzyme indicators based on fluorophores such as 7-hydroxycoumarines (soluble fluorogenic indicators) or quinazolines (precipitating fluorogenic indicators—see for instance: EP 0 641 351 A1) lack significant absorption in the visible electromagnetic band and thus escape detection by the human eye without the application of optical instrumentation for interrogation. In contrast, common chromogenic indicators such those derived from 3-indoxyls lack fluorescence. This is true in particular for precipitating indicators since fluorescence in the solid state is a rare phenomenon (due to the well known effect of self-quenching of excited molecules arranged in a tight lattice).
It would be advantageous over the current state of the art if enzyme indicators were made available that have both chromogenic and fluorogenic properties.
Diazonium Staining
In histology, another type of indicator is well known and often used to localize enzyme activity. This important method was pioneered by Seligman et al. (J. Histochem. Cytochem. 1954, 2, 209-229), Burstone et al. (J. Histochem. Cytochem. 1956, 4, 217-226) and Rutenburg et al. (J. Histochem. Cytochem. 1958, 6, 122-129). It is based on the reaction of stabilized diazonium salts with electron rich aromatic amines and phenols to form azo dyes. Many azo dyes are of intense color and some are fluorescent.
This diazonium coupling reaction will proceed with aromatic amines and phenols much faster than with their corresponding esters and amides. Therefore, hydrolytic enzyme activity can be detected by exposing a sample to a suitable phenolic ester or anilide followed by staining with diazonium salts. Depending on the substrate and the diazonium salts, good localization can be achieved.
In practice, different types of chromogens such as naphtylamines or naphthol derivatives are employed. There exists considerable variety of commercially available substrates of this type along with suitable diazonium staining salts.
Pearson et al. (Proc. Soc. Exptl. Biol. Med. 1961, 108, 619-613; Lab. Invest. 1963, 12, 712-720), Yarborough et al. (J. Reticuloendothelial Soc. 1967, 4, 390-408), Gossrau et al. (Histochemistry, 1987, 397-404) and others developed staining methods that are based on the reaction of Indox indicators with diazonium electrophiles for use in histology.
Mohler and Blau (Proc. Natl. Acad. Sci. USA 1996, 12423-12427) have evaluated many indicator systems for beta-D-galactosidase combining Indox indicators and various commercial diazonium salts.
It should be noted, however, that due to the carcinogenicity and high toxicity towards humans and cell cultures and the otherwise hazardous nature of diazonium salts this method poses significant danger to the user and is not compatible with either in vivo or non-destructive type of assaying in general.
In view of all the above, a superior type of indicator releasing activated signalogen which spontaneously yield signalophores in a process that is entirely independent of external factors such as molecular oxygen or any reagent or chemical species present in the surrounding environment would be desirable.
Metal Chelates
Another class of precipitating substrates are based on metal chelating molecules that form insoluble complexes with metal ions. Chelating molecules contain two or more functional groups that coordinate to a metal ion. These functional groups can be masked by labile groups to prevent the formation of a metal complex which represents a relatively common design of indicator.
Enzyme indicators based on metal chelates do have some practical relevance. For example, substrates derived from 8-hydroxyquinolines such as 8-hydroxyquinoline-beta-D-glucuronide or the naturally occurring esculetin (esculin-beta-D-glucopyranoside) can be used to detect the corresponding enzymes in the presence of iron(III) salts.
However, these indicators suffer from other drawbacks: Many of these chelating molecules display biocidal effects on culture. Such toxicity is intrinsic to any metal chelating agents since they generally interfere with the functioning of metallo-enzymes. Further, one must maintain a certain concentration of metal ions present in the assay, which may cause undesirable interference. In addition, most stains produced from metal chelation are brown and hence will not provide good contrast from background.
For example, 8-hydroxyquinoline is an excellent ligand that forms stable complexes with many transition metals. Masking the hydroxyl group with a suitable labile group produces potentially useful indicators. The assay will release 8-hydroxyquinoline as the activated signalogen which rapidly binds iron(II or III) thereby forming a precipitating metal complex that is dark colored. Moreover, 8-hydroxyquinoline possesses significant anti-microbial activity which may inhibit the growth of some prominent organisms within the format of a microbial assay. Accordingly, enzyme indicators preferably should not involve chelating agents or unnatural metal ion concentrations which by nature interfere with the course of the assay.